This project has grown to include five groups: the NTLF, the Lipscomb group at the University of Minnesota, the Floss group at the University of Washington, the Lippard group at MIT, and the Chan group at CalTech. Methanotrophic bacteria play an essential part in cycling carbon in the biosphere by consuming methane produced in anaerobic sediments and by limiting its flux to the atmosphere where it acts as a greenhouse gas. The first step in this process is catalyzed by methane monooxygenase (MMO) systems, which convert methane to methanol. For several methanotrophs, the methane monooxygenase enzyme complex consists of three essential proteins: a 245 kDa hydroxylase component (MMOH), a 40 kDa NAD(P)H dependent oxidoreductase protein, and a 15.8 kDa component B which has no associated co-factors. Both the Lipscomb and Lippard groups have investigated the structure and mechanism of the MMOH catalytic center, and the interaction of the other components with MMOH, by a wide variety of physical and spectroscopic techniques, including Mossbauer and EPR studies, ENDOR, optical, CD and Raman spectroscopy. Recently it has been shown that the product distribution (i.e. the ratio of primary vs secondary alcohol products) can be affected by the quantity of Component B or Reductase present in the NADH and O2-coupled oxidation reaction. Our initial collaborative efforts with the Lipscomb and Floss groups on the proteins from the Methylosinus trichosporium OB3b bacterium have been described previously. Briefly, we synthesized multi-Curie amounts of enantiomerically pure (R)- and (S)-[1-2H1, 3H1]ethane and used these labelled ethanes as substrates for both the fully reconstituted MMO coupled to NADH/O2, and for the MMOH/H2O2 system. Analysis of the ethanol products, and of the mandelate derivatives of the alcohols by 3H NMR spectroscopic techniques showed that, regardless of whether NADH/O2 or H2O2 are the source of oxygen and reducing equivalents, the hydroxylation of ethane proceeds with the same steric course, predominantly retention of configuration. Recent questions we have addressed include: (i). does the temperature of incubation affect the retention vs inversion ratio for the simple ethane substrate, and can this result give us information about the reaction intermediate? (ii). does the bulkiness of the substrate affect the retention vs inversion ratio in the oxidized alcohol? (iii). how much mobility does the substrate have in the active site of the enzyme? (iv). if the product distributions vary with Component B stoichiometry, do we still observe the same retention vs inversion of configuration at carbon? We have built on the extensive studies of product distributions already compiled by the Lipscomb group (using GLC and HPLC analyses) and chose butane as our "larger molecule" for the second series of collaborative studies. Since the products could be oxidized at the primary or secondary carbon atoms, it was necessary to synthesize both primary and secondary chirally tritiated butanes, and the tosyl precursors for these tritiation experiments were prepared by the Floss group. Recently we began a collaboration with the Lippard group (MIT), using their MMO derived from Methylococcus capsulatis (Bath). We performed experiments with selected substrates to investigate the reported differences in catalytic behavior of the MMO enzyme complexes from the two methanotrophs. Our preliminary studies gave significantly different results, and subsequent repeat experiments have been very interesting. The latest series of incubations (using highly purified enzyme) have given essentially the same results as the Methylosinus trichosporium OB3b enzyme system. Early in 1995 this project was extended further by a collaboration with the Chan group from California Institute of Technology. This phase of the project has the added interest that the enzyme is membrane-bound, and is Copper-containing (as distinct from the isolated, purified, iron-containing enzymes from both the U. Minnesota and MIT groups). As might be expected, the stereochemical results obtained from digestion experiments using this enzyme system are completely different to our previous observations. We are currently evaluating these results, and planning future studies to confirm and build on the unique results obtained so far. After confirmatory experiments during late 1995, the CalTech research was published in J. Am. Chem. Soc. Most experimental work on these projects has been completed, and considerable results need to be published from work with the Minnesota and MIT enzymes.